Fig. 1. Validation of the assembly and activity of Loaded-Enc and Empty-Enc nanocompartments.

(A) SDS–polyacrylamide gel electrophoresis (PAGE) of purified Empty-Enc and Loaded-Enc. Proteins are resolved by 15% acrylamide SDS-PAGE and stained with Coomassie blue stain. Encapsulin bands are near the 30-kDa marker and are highlighted by a black arrow. The EncFtn cargo of Loaded-Enc appears as both a monomer and a dimer. Bands corresponding to the monomer and dimer of EncFtn are highlighted with green arrows. (B) Recombinant, purified Empty-Enc (blue trace) and Loaded-Enc (orange, dotted trace) were analyzed by SEC using a Sephacryl 400 column (Cytiva). Both encapsulins elute at the same volume, suggesting that the encapsulin complexes are the same size regardless of cargo loading. (C) Negative-stain transmission micrographs of Empty-Enc (upper micrograph) and Loaded-Enc (lower micrograph) displaying individual particles for each complex. One nanocompartment of Empty-Enc and Loaded-Enc is shown in the upper right corner of each micrograph, with a hexagonal 2D geometry observed. (D) Top: Ferroxidase activity of Loaded-Enc compared to Empty-Enc. Protein samples were mixed with 100 μM FeSO₄.7H₂O. Following an incubation period at room temperature of 50 s, absorbance at 315 nm was measured over a time course of 1450 s. Control reference established using enzyme-free reaction as a measure of background iron oxidation. The lines represent the mean of all repeats; error bars represent the SD from the mean. Bottom: End point ferroxidase assay comparison. Ferroxidase activity shown by the total increase in A₃₁₅ (absorbance at 315 nm) at the end point of the assay. Bars represent the mean of all repeats; error bars represent the SD from the mean. AU, Absorbance Units.